POS0426 BRD3 REGULATES THE INFLAMMATORY AND STRESS RESPONSE IN RHEUMATOID ARTHRITIS SYNOVIAL FIBROBLASTS
Authors: M. Maciukiewicz, L. Moser, M. Krosel, T. Seifritz, M. Tomsic, B. Maurer, O. Distler, C. Ospelt, K. Klein
Journal: Annals of the Rheumatic Diseases (2022)

Background Small molecule inhibitors targeting members of the bromodomain and extra-terminal (BET) protein family (BRD2, BRD3, BRD4) have anti-inflammatory properties in rheumatoid arthritis (RA). BET proteins are readers of acetylated histone side chains and activators of transcription. BRD3 is an understudied member of BET proteins. Objectives To analyse individual functions of BET proteins and mechanisms underlying BET inhibition in RA synovial fibroblasts (SF). Methods The expression of BRD2, BRD3, and BRD4 was silenced by lenti-viral transduction followed by TNF stimulation (10 ng/µl, 24h). Silencing was confirmed by Western blotting. Transcriptomes were determined by RNA-seq (Illumina NovaSeq 6000, n=3). Pathway enrichment analysis for KEGG and Reactome databases was conducted with significantly affected genes (± fold change &gt; 1.5, FDR &lt; 0.05). SF were treated with I-BET (1 µM) and TNF (10 ng/µl, 24h). Autophagy was evaluated by Western blotting using the conversion of LC3B as a marker (n=9). I-BET-induced global changes on post-translational histone modifications were analysed by mass spectrometry (Mod Spec, Active Motif; n=2; 120h protocol) and Western blotting (H3K27ac, H3K18ac, total acH3; n=7; 24h and 120h protocol). For this purpose, SF were stimulated with I-BET (1 µM) for 24h, and either co-stimulated with TNF (24h protocol), or washed with PBS, followed by a 24h stimulation with TNF 120h after the I-BET treatment (120h protocol). Results Silencing of BRD2 and BRD4 in SF was, in contrast to silencing of BRD3, associated with high levels of cell death, and therefore not analyzed further. We detected 257 and 324 differentially expressed genes (DEG) that were affected by BRD3 silencing in unstimulated and TNF-stimulated SF, respectively. 105 DEG overlapped between the two groups. DEG were enriched in inflammatory pathways such as “TNF signaling pathway”, “rheumatoid arthritis”, “Toll-like receptor cascades”, “MAPK signaling pathway”, “IL-17 signaling pathway” and “signaling by interleukins”. Furthermore, pathway enrichment analysis suggested a role for BRD3 in different stress-associated pathways, including “DNA repair”, “chaperone mediated autophagy”, “cellular responses to stress”, and “autophagy”. In line with the pathway enrichment analysis, I-BET induced levels of LC3B-II in unstimulated (4.3 fold, p=0.07) and TNF-stimulated (2.9 fold, p=0.07) SF, indicating a role of BET proteins in the regulation of autophagy. To further study the mechanisms underlying I-BET-mediated suppression of gene expression, we analyzed potential effects of I-BET on histone modifications. Mod Spec analysis indicated that I-BET induced profound changes in chromatin modifications, with a global reduction of acetylation on different histone side chains. We confirmed some of these differences in independent samples. I-BET treatment reduced mean TNF-induced levels of total acH3 by 25.2% (120h; p=0.0303), of H3K18ac by 35.3% (24h; p=0.0288) and by 29.3% (120h; p=0.0373) and of H3K27ac by 41.7% (120h; p=0.0587). Conclusion BRD3 acts as an upstream regulatory factor that integrates the response to inflammatory stimuli and stress conditions in SF. Our data suggest that BET inhibitors do not only prevent the reading of acetylated histone side chains, but also directly affect the chromatin structure, in particular by downregulating global levels of histone acetylation. Disclosure of Interests Malgorzata Maciukiewicz: None declared, Larissa Moser: None declared, Monika Krosel: None declared, Tanja Seifritz: None declared, Matija Tomsic: None declared, Britta Maurer Speakers bureau: Boehringer-Ingelheim, Consultant of: Novartis, Boehringer Ingelheim, Janssen-Cilag, Grant/research support from: AbbVie, Protagen, Novartis Biomedical Research, Oliver Distler Consultant of: Abbvie, Caroline Ospelt: None declared, Kerstin Klein Grant/research support from: Novartis Foundation for biomedical research (2019)